In our study on the chemistry of 4-hydroxy-2-nonenal (HNE), a cytotoxic aldehyde produced by lipid peroxidation, we observed the presence of a distinct fluorescent component among the products from the reaction of HNE with acetyllysine. This fluorophore exhibited spectral properties similar to those described for the age-pigment, lipofuscin. Mass spectroscopic analysis revealed a composition consistent with a compound derived from an oxidative condensation of two molecules of acetyllysine with one molecule of HNE. We have established the generality of this reaction by demonstrating the production of analogous fluorophore from the action of HNE with various amino compounds, viz.: gamma-aminobutyric acid, beta-alanine, and their ethyl esters and ethylamine. The reaction with ethylamine is of particular interest since it is probably the smallest amine to furnish a fluorophore upon reaction with HNE. The mass spectral data of this fluorophore showed a fragmentation peak corresponding to the elimination of a molecule of ethylamine, thus indicating a chromophore of only five carbon and one nitrogen atoms. However, due to the very low yield of this reaction, the data at present are not amenable to a definitive assignment of structure. Polyclonal antibodies were raised against a purified sample of acetyllysine-HNE fluorophore. Thus, an antibody specific to the fluorophore was obtained by affinity chromatography. This antibody was shown to be highly specific towards the lysine-HNE fluorophore and exhibited no cross-reactivity towards 1:1 cysteine and histidine-HNE adducts and only very low reactivity towards non-fluorescent 1:1 and 2:1 lysine-HNE adducts. The antibody recognized lysine-HNE fluorophore on HNE-treated glucose-6-phosphate dehydrogenase and in rat brain tissue. Hence, this antibody can be valuable as a specific biological marker for lipofuscin derived from lysine residues in proteins.